The introduction of therapeutic genes into cells for the treatment of diseases as diverse as those resulting from genetic defects, cancer and viral infections is the major aim of gene therapy. Cancer and diseases such as AIDS resulting from infection with human immunodeficiency virus (HIV) are particularly difficult to treat even though a number of clinical protocols are presently underway that use gene therapeutical approaches. The amphipathic peptide melittin, the major component of bee venom, has been shown to have selective anti-cancer (Sharma, S. V., Oncogene, 7:193–201 (1992); Sharma, S. V., Oncogene, 8:939–947 (1993)) and anti-HIV activity (Wachinger et al., FEBS Lett., 309:235–241 (1992)); U.S. Pat. No. 4,822,608 of Benton et al. and WO patent application 91/08753 of Erfle et al. both relate to these therapeutic properties.
U.S. Pat. No. 4,822,608 issued to Benton et al. on Apr. 18, 1989 and entitled “METHODS AND COMPOSITIONS FOR THE TREATMENT OF MAMMALIAN INFECTIONS EMPLOYING MEDICAMENTS COMPRISING HYMENOPTERA VENOM OR PROTEINACEOUS OR POLYPEPTIDE COMPONENTS THEREOF” teaches that secondary agents derived from nature such as hymenoptera venom or proteinaceous or polypeptide components thereof has a potentiating effect on antibacterial agents. This reference further suggests that such compositions may also have increased anti-viral, carcinostatic and anti-carcinogenic effects on various maladies. More particularly, the reference to Benton et al. discloses the use of melittin which is the main component of honey bee toxin, in combination with assorted antibiotic agent as having antibacterial activity against predetermined infections. Further this reference teaches that a synergistic benefit may be achieved by the combination of the melittin and assorted antibiotics in various therapeutically effective amounts.
WO patent application 91/08753 of Erfle et al. relates to a method and composition for the treatment of mammalian HIV infections, and more particularly to such a method and composition for treating mammalian HIV infections which employs hymenoptera venom, or proteinaceous or polypeptide components thereof and which is introduced into the mammalian hosts and which are individually operable to restrict or substantially inhibit the virus replication in the HIV infected cells of the mammal.
In these studies purified melittin peptide was given to cells in culture which, though useful for experimental purposes, is not relevant for therapy. Even in vivo administration of purified melittin protein (for example i.v.) is probably not advisable because of the relatively high concentrations and repeated doses that would be required to maintain therapeutic levels. Further, since this kind of generalized delivery would result in the amphipathic peptide reaching not only target cells but also other cells, thereby potentially resulting in nondesirable side effects, it would be advantageous to be able to target the delivery of melittin or other antimicrobial peptides, in particular the melittin peptide and the peptides mentioned below.
A second class of therapeutic genes of interest are the cecropins isolated from the pupae of giant silk moths (Bowman, H. G., Ann. Rev. Immunol., 13:1–51 (1995), Hultmark et al., Eur. J. Biochem., 106:7–16 (1980)). There are three principal cecropins, A, B and D with a similar structure to melittin (Hultmark et al., Eur. J. Biochem., 127:207–217 (1982)). Cecropins A and B show specific antibacterial activity without any apparent ill effects for mammalian cells (Steiner H. et al., Nature, 292:246–248 (1991)). Recently Moore and coworkers have shown that the cecropin B, P and Shiva-1 antibacterial peptides show anticancer activity against a variety of tumour cell lines (Moore, A. J. et al., Peptide Research, 7:265–269 (1994)).
Cecropins were first isolated from the hemolymph of the giant silk moth, Hyalophora cecropia, following induction by live non-pathogenic bacteria. The principal insect cecropins (A, B and D) are 35 to 37 residues long, devoid of cystein and have a strongly basic N-terminus linked to a neutral C-terminius by a flexible glycine-proline link. The overall structure deduced by NMR for cecropin A is two nearly perfect amphipathic segments joined by a Gly-Pro hing. A cecropin-like 31-residue peptide (cecropin P), isolated from the small intestine of a pig (Lee et al., Proc. Natl. Acad. Sci. USA, 86:9159–9162 (1989)), suggests that the cecropins may be widespread throughout the animal kingdom. The mechanism of action of the cecropins is thought to involve channel formation in membranes and subsequent lysis.
SB-37 (a close cecropin B analogue) and Shiva-1 (a cecropin B analogue that shares about 40% sequence homology and maintains the same charge distribution and hydrophobicity as the peptide) have been shown to lyse several mammalian leukemia and lymphoma cell lines in vitro. The publication of Moore, A. J. et al., Peptide Research, 7:265–269 (1994) is incorporated herein by reference for complete disclosure. Similar antitumour effects have been demonstrated for the magainins, a related group of antimicrobial peptides (Cruciani, R. A. et al., Proc. Natl. Acad. Sci. USA, 88:3792–3796 (1991); Ohaski, Y. et al., Cancer Research, 52:3534–3538, (1992)).
The use of retroviral vectors (RV) for gene therapy has received much attention and currently is the method of choice for the transferral of therapeutic genes in a variety of approved protocols both in the USA and in Europe (Kotani, H. et al., Human Gene Therapy, 5:19–28 (1994)). However most of these protocols require that the infection of target cells with the RV carrying the therapeutic gene occurs in vitro, and successfully infected cells are then returned to the affected individual (Rosenberg, S. A. et al., Human Gene Therapy, 3:75–90 (1992); for a review see Anderson, W. F., Science, 256:808–813 (1992)). Such ex vivo gene therapy protocols are ideal for correction of medical conditions in which the target cell population can be easily isolated (e.g. lymphocytes). Additionally the ex vivo infection of target cells allows the administration of large quantities of concentrated virus which can be rigorously safety tested before use.
Unfortunately, only a fraction of the possible applications for gene therapy involve target cells that can be easily isolated, cultured and then reintroduced. Additionally, the complex technology and associated high costs of ex vivo gene therapy effectively preclude its disseminated use world-wide. Future facile and cost-effective gene therapy will require an in vivo approach in which the viral vector, or cells producing the viral vector, are directly administered to the patient in the form of an injection or simple implantation of RV producing cells.
This kind of in vivo approach, of course, introduces a variety of new problems. First of all, and above all, safety considerations have to be addressed. Virus will be produced, possibly from an implantation of virus producing cells, and there will be no opportunity to precheck the produced virus. It is important to be aware of the finite risk involved in the use of such systems, as well as trying to produce new systems that minimize this risk.
The essentially random integration of the proviral form of the retroviral genome into the genome of the infected cell led to the identification of a number of cellular proto-oncogenes by virtue of their insertional activation (Varmus, H., Science, 240:1427–1435 (1988)). The possibility that a similar mechanism may cause cancers in patients treated with RVs carrying therapeutic genes intended to treat other pre-existent medical conditions, has posed a recurring ethical problem. Most researchers would agree that the probability of a replication defective RV, such as all those currently used, integrating into or near a cellular gene involved in controlling cell proliferation is vanishingly small. However, it is generally also assumed that the explosive expansion of a population of replication competent retrovirus from a single infection event, will eventually provide enough integration events to make such a phenotypic integration a very real possibility.
Retroviral vector systems are optimized to minimize the chance of replication competent virus being present. However, it has been well documented that recombination events between components of the RV system can lead to the generation of potentially pathogenic replication competent virus and a number of generations of vector systems have been constructed to minimize this risk of recombination (reviewed in Salmons, B. and Günzburg, W. H., Human Gene Therapy, 4:129–141 (1993)). However little is known about the finite probability of these events. Since it will never be possible to reduce the risk associated with this or other viral vector systems to zero, an informed risk-benefit decision will always have to be taken. Thus it becomes very important to empirically determine the chance of (1) insertional disruption or activation of single genes by retrovirus integration and (2) the risk of generation of replication competent virus by recombination in current generations of packaging cell lines. A detailed examination of the mechanism by which these events occur will also allow the construction of new types of systems designed to limit these events.
A further consideration for practical in vivo gene therapy, both from safety considerations as well as from an efficiency and from a purely practical point of view, is the targeting of RVs. It is clear that therapeutic genes carried by vectors should not be indiscriminately expressed in all tissues and cells, but rather only in the requisite target cell. This is especially important if the genes to be transferred are toxin genes aimed at ablating specific tumour cells. Ablation of other, nontarget cells would obviously be very undesirable. Targeting of the expression of carried therapeutic genes can be achieved by a variety of means.
Retroviral vector systems consist of two components (FIG. 1):                1) the retroviral vector itself is a modified retrovirus (vector plasmid) in which the genes encoding for the viral proteins have been replaced by therapeutic genes optionally including marker genes to be transferred to the target cell. Since the replacement of the genes encoding for the viral proteins effectively cripples the virus it must be rescued by the second component in the system which provides the missing viral proteins to the modified retrovirus.        
The second component is:                2) a cell line that produces large quantities of the viral proteins, however lacks the ability to produce replication competent virus. This cell line is known as the packaging cell line and consists of a cell line transfected with a second plasmid carrying the genes enabling the modified retroviral vector to be packaged. This plasmid directs the synthesis of the necessary viral proteins required for virion production.        
To generate the packaged vector, the vector plasmid is transfected into the packaging cell line. Under these conditions the modified retroviral genome including the inserted therapeutic and optional marker genes is transcribed from the vector plasmid and packaged into the modified retroviral particles (recombinant viral particles). A cell infected with such a recombinant viral particle cannot produce new vector virus since no viral proteins are present in these cells. However the vector carrying the therapeutic and marker genes is present and these can now be expressed in the infected cell.